Methods and compositions for modulation of migration of neurogenic cells

ABSTRACT

Disclosed herein are methods for the treatment of traumatic brain injury by transplantation of cells descended from marrow adherent stem cells that express an exogenous Notch intracellular domain. The transplanted cells form a pathway along which endogenous neurogenic cells proliferate and migrate from the subventricular zone to the site of injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/285,700 filed Feb. 26, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/489,934 filed Sep. 18, 2014, which is acontinuation of U.S. patent application Ser. No. 13/800,585 filed Mar.13, 2013 (U.S. Pat. No. 9,828,593 issued Nov. 28, 2017), which claimsthe benefit of U.S. Provisional Patent Application No. 61/647,893 filedMay 16, 2012, the contents of which are incorporated herein by referencein their entireties.

STATEMENT REGARDING FEDERAL SUPPORT

Some of the research described herein was supported by a grant from theNational Institute of Neurological Disorders and Stroke. The UnitedStates government may have certain rights in the inventions disclosedherein.

FIELD

The present disclosure is in the field of therapy for neurologicaldisorders.

BACKGROUND

Initially employed for in-depth examination of cell development¹, stemcells have become a cornerstone for regenerative medicine, includingcell-based therapies for treatment of neurological disorders^(2,3). Stemcells exist even in adulthood⁸, and possess the capacity to self-renewand differentiate into multiple lineages⁹, contribute to normalhomeostasis¹⁰, and exert therapeutic benefits either endogenously¹¹⁻¹⁴or following transplantation in injured organs, i.e., brain¹⁵⁻²¹. Thesubventricular zone (SVZ) of the lateral ventricles and the sub granularzone of the hippocampus dentate gyrus are the two major stem-cell nichesin the adult brain^(22,23), although quiescent neural stem cells (NSCs)have been detected in other brain regions²⁴. Induction of endogenousstem cells after injury would provide new opportunities in regenerativemedicine^(2,3,11-21).

Cells other than pluripotent stem cells have also been used in thetreatment of disorders of the central nervous system. As one example,SB623 cells (which are cells derived from marrow adherent stem cells inwhich an exogenous Notch intracellular domain has been expressed) areused for the treatment of stroke, by transplantation at or near the siteof ischemic insult. See, for example, U.S. Pat. No. 8,092,792 andYasuhara et al. (2009) Stem Cells Devel. 18:1501-1513. U.S. Pat. No.7,682,825 describes additional uses of SB623 cells in the treatment of anumber of disorders of the central and peripheral nervous systems.

Despite these scientific advances and some initial clinicalstudies²⁵⁻²⁷, a fundamental gap in our understanding of cell therapy isa knowledge of the mechanisms by which transplanted cells facilitate therepair of damaged neural tissue. To date, increased graft survival andgraft persistence have been considered the crux of successful celltransplantation therapy in affording therapeutic benefits in hematologicand non-hematologic disorders. Thus, much effort has been directed toprolonging the survival and persistence of transplanted cells.Accordingly, methods for effective cell therapy, that do not require thepersistence of large amounts of transplanted cells, would beadvantageous.

Traumatic brain injury (TBI) refers to damage to the brain resultingfrom external mechanical force. TBI can result from falls, firearmwounds, sports accidents, construction accidents and vehicle accidents,among other causes. Victims of TBI can suffer from a number of physical,cognitive, social, emotional and/or behavioral disorders.

Little can be done to reverse the initial physical damage of a TBI.Therefore, treatment options consist primarily of stabilization toprevent further damage in the acute phase, and rehabilitationthereafter. Because of these limited options, additional methods andcompositions for treatment of TBI are needed.

SUMMARY

The present inventors have discovered that transplantation of SB623cells (i.e., cells derived from marrow adherent stem cells in which anexogenous Notch intracellular domain has been expressed) can be used inthe treatment of traumatic brain injury (TBI). Animals that receivedtransplants of SB623 cells after TBI displayed significantly improvedmotor and neurological functions coupled with significantly reduceddamage to the cortical core and peri-injured cortical areas, compared totraumatically injured animals that received injection of vehicle only.

The inventors have also found that, contrary to expectations, survivaland persistence of large numbers of transplanted cells are not requiredfor the therapeutic benefits of SB623 cell transplantation.Surprisingly, therapeutic benefits can be obtained by minimum and acutegraft survival, which is sufficient to initiate a robust and stablefunctional recovery. This solves two major problems: the need for anample supply of transplantable cells and the need for long-term graftsurvival.

The inventors have also discovered that the beneficial effects of SB623cell transplantation, in the treatment of TBI, result from the formationof a biological bridge (“biobridge”) between the neurogenic niche in thesubventricular zone (SVZ) and the injured brain site. This biobridge,which has been visualized immunohistochemically and laser-captured,initially expressed high levels of extracellular matrixmetalloproteinases and was characterized by a stream of the transplantedcells. At later times after transplantation, the grafted cells werereplaced by newly formed host cells, and few-to-no transplanted cellsremained in the biobridge. Thus, the transplanted SB623 cells initiallyformed a pathway between the neurogenic SVZ and the injured cortex thatfacilitated later migration of host neurogenic cells from the neurogenicniche to the site of brain injury.

This sequence of events reveals a novel method for treatment of TBI;namely, transplantation of SB623 cells, which form transient pathwaysfor directing the migration of host neurogenic cells. That is, thetransplanted SB623 cells initially form a biobridge between a neurogenicniche and the site of injury; but once this biobridge is formed, thegrafted cells are replaced by host neurogenic cells which migrate to theinjury site. These findings reveal that long-distance migration of hostcells from a neurogenic niche to an injured brain site can be achievedthrough transplanted SB623 cells serving as biobridges for initiation ofendogenous repair mechanisms.

Accordingly, the present disclosure provides, inter alia, the followingembodiments:

1. A method for treating traumatic brain injury in a subject, the methodcomprising administering, to the brain of the subject, a therapeuticallyeffective amount of SB623 cells, wherein the SB623 cells are obtained by(a) providing a culture of marrow adherent stem cells (MSCs), (b)contacting the cell culture of step (a) with a polynucleotide comprisingsequences encoding a Notch intracellular domain (NICD) wherein saidpolynucleotide does not encode a full-length Notch protein, (c)selecting cells that comprise the polynucleotide of step (b), and (d)further culturing the selected cells of step (c) in the absence ofselection.

2. The method of embodiment 1, wherein the subject is a human.

3. The method of either of embodiments 1 or 2, wherein the MSCs areobtained from a human.

4. Cells for transplantation into a subject for the treatment oftraumatic brain injury, wherein said cells are obtained by a processcomprising the steps of: (a) providing a culture of MSCs, (b) contactingthe cell culture of step (a) with a polynucleotide comprising sequencesencoding a NICD wherein said polynucleotide does not encode afull-length Notch protein, (c) selecting cells that comprise thepolynucleotide of step (b), and (d) further culturing the selected cellsof step (c) in the absence of selection.

5. The cells of embodiment 4, wherein the subject is a human.

6. The cells of either of embodiments 4 or 5, wherein the MSCs areobtained from a human.

7. A method for inducing the migration of endogenous neurogenic cellsfrom a neurogenic niche to a site of brain injury, the method comprisingadministering, to the brain of a subject, a therapeutically effectiveamount of SB623 cells, wherein the SB623 cells are obtained by (a)providing a culture of MSCs, (b) contacting the cell culture of step (a)with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein, (c) selecting cells that comprise thepolynucleotide of step (b), and (d) further culturing the selected cellsof step (c) in the absence of selection.

8. The method of embodiment 7, wherein the neurogenic niche is thesubventricular zone.

9. The method of either of embodiments 7 or 8, wherein the brain injuryis a traumatic brain injury.

10. The method of any of embodiments 7-9, wherein the subject is ahuman.

11. The method of any of embodiments 7-10, wherein the MSCs are obtainedfrom a human.

12. A method for stimulating proliferation of neurogenic cells in asubject, the method comprising administering, to the brain of a subject,a therapeutically effective amount of SB623 cells, wherein the SB623cells are obtained by (a) providing a culture of MSCs, (b) contactingthe cell culture of step (a) with a polynucleotide comprising sequencesencoding a Notch intracellular domain (NICD) wherein said polynucleotidedoes not encode a full-length Notch protein, (c) selecting cells thatcomprise the polynucleotide of step (b), and (d) further culturing theselected cells of step (c) in the absence of selection.

13. The method of embodiment 12, wherein the brain injury is a traumaticbrain injury.

14. The method of either of embodiments 12 or 13, wherein the subject isa human.

15. The method of any of embodiments 12-14, wherein the MSCs areobtained from a human.

16. A method for inducing neurogenic cells to proliferate and migrate toa site of brain injury in a subject, the method comprisingadministering, to the brain of a subject, a therapeutically effectiveamount of SB623 cells, wherein the SB623 cells are obtained by (a)providing a culture of MSCs, (b) contacting the cell culture of step (a)with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD) wherein said polynucleotide does not encodea full-length Notch protein, (c) selecting cells that comprise thepolynucleotide of step (b), and (d) further culturing the selected cellsof step (c) in the absence of selection.

17. The method of embodiment 16, wherein the brain injury is a traumaticbrain injury.

18. The method of either of embodiments 16 or 17, wherein the subject isa human.

19. The method of any of embodiments 16-18, wherein the MSCs areobtained from a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of the elevated body swing test (EBST) in rats.Values are provided for Baseline (before TBI) and for 7 days, 1 month, 2months and 3 months after TBI. The rats received either transplants ofSB623 cells (“Cells,” right-most bar in each pair) or infusion ofvehicle (“Vehicle,” left-most bar in each pair). “*” indicatesstatistical significance with a p<0.05.

FIG. 2 shows mean scores in a modified Bederson neurological examinationin rats subjected to TBI that subsequently received either transplantsof SB623 cells (“Cells”) or infusion of vehicle (“Vehicle”). Values areprovided for Baseline (before TBI) and for 7 days, 1 month, 2 months and3 months after TBI. The left-most bar in each pair represents the scorein rats infused with vehicle; the right-most bar in each pair representsthe score in rats that received transplants of SB623 cells. “*”indicates statistical significance with a p<0.05.

FIG. 3 shows mean values for the number of seconds rats were able toremain on a Rotorod apparatus. The rats were subjected to experimentalTBI and subsequently received either transplants of SB623 cells(“Cells”) or infusion of vehicle (“Vehicle”). Values are provided forBaseline (before TBI) and for 7 days, 1 month, 2 months and 3 monthsafter TBI. The left-most bar in each pair represents the score in ratsinfused with vehicle; the right-most bar in each pair represents thescore in rats that received transplants of SB623 cells. “*” indicatesstatistical significance with a p<0.05.

FIGS. 4A and 4B shows results of assays for damage to the cortical core(“Core”) and to the cortical region in and around the impact site(“Peri-injury”) in rats subjected to TBI. FIG. 4A shows H&E sections ofbrains from rats that received transplants of SB623 cells (panels a-d)compared to rats that received infusion of vehicle (panels a1-d1). InFIG. 4B, the results are expressed as percent lesioned area (see Example8) relative to animals subjected to TBI that received infusion ofvehicle. The left-most bar in each pair shows values for the coreregion; the right-most bar in each pair shows values for the peri-injuryregion.

FIG. 5 shows levels of Ki67-labeled cells in the subventricular zone(“SVZ”) and the cortex (“CTX”) of animals subjected to TBI that receivedtransplants of SB623 cells, compared to animals subjected to TBI thatreceived infusions of vehicle, at one month and three months after TBI.“*” indicates a statistically significant increase in the number oflabeled cell observed per high-power field (p<0.05).

FIG. 6 shows levels of nestin-labeled cells in the subventricular zone(“SVZ”) and the cortex (“CTX”) of animals subjected to TBI that receivedtransplants of SB623 cells, compared to animals subjected to TBI thatreceived infusions of vehicle, at one month and three months after TBI.“*” indicates a statistically significant increase in the number oflabeled cell observed per high-power field (p<0.05).

FIG. 7 shows levels of doublecortin-labeled cells in the corpus callosum(“CC”) and the cortex (“CTX”) of animals subjected to TBI that receivedtransplants of SB623 cells, compared to animals subjected to TBI thatreceived infusions of vehicle, at one month and three months after TBI.“*” indicates a statistically significant increase in the number oflabeled cell observed per high-power field (p<0.05).

FIG. 8 shows lytic activity in homogenates of laser-captured biobridgesfrom the brains of rats subjected to experimental TBI, at one month andthree months after TBI. Activities are expressed as optical densityunits, relative to 0.5 ng recombinant MMP-9, obtained by scanningzymographic gels. In each of the two sets of three bars, the left-mostbar represents relative activity in biobridges from rats infused withvehicle after TBI, the center bar represents relative activity inbiobridges from rats transplanted with SB623 cells after TBI, and theright-most bar represents relative activity in biobridges from control,sham-operated rats.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions for treatment of traumaticbrain injury (TBI). Also disclosed herein are methods and compositionsfor modulation of the migration of stem cells (e.g., neural stem cells,neuronal stem cells) in the brain.

The inventors have made the surprising discovery that the behavioral andhistological improvements resulting from cell transplantation, afterTBI, do not require large-scale graft survival or long-term graftpersistence. Indeed, only modest, acute graft survival is necessary toproduce these therapeutic benefits. Thus, the inventors have uncovered anovel method for neural repair that entails a threshold dose oftransplanted cells, which do not need to persist in the brain, and whichare capable of inducing the SVZ to generate and propel new cells to theimpacted cortical area. Accordingly, transplanting the minimum effectivedose and the acute survival of the transplanted cells are sufficient toinitiate an intricate endogenous restorative machinery for abrogating amassive brain injury.

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields of cellbiology, toxicology, molecular biology, biochemistry, cell culture,immunology, neurology, surgery, recombinant DNA and related fields asare within the skill of the art. Such techniques are described in theliterature and thereby available to those of skill in the art. See, forexample, Alberts, B. et al., “Molecular Biology of the Cell,” 5^(th)edition, Garland Science, New York, N.Y., 2008; Voet, D. et al.“Fundamentals of Biochemistry: Life at the Molecular Level,” 3^(rd)edition, John Wiley & Sons, Hoboken, N.J., 2008; Sambrook, J. et al.,“Molecular Cloning: A Laboratory Manual,” 3rd edition, Cold SpringHarbor Laboratory Press, 2001; Ausubel, F. et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, New York, 1987 and periodicupdates; Freshney, R. I., “Culture of Animal Cells: A Manual of BasicTechnique,” 4th edition, John Wiley & Sons, Somerset, N.J., 2000; andthe series “Methods in Enzymology,” Academic Press, San Diego, Calif.

Marrow Adherent Stem Cells (MSCs)

The present disclosure provides methods for treating TBI and modulatingstem cell migration by transplanting SB623 cells to a site of braininjury in a subject. SB623 cells are obtained from marrow adherent stemcells (MSCs), also known as marrow adherent stromal cells andmesenchymal stem cells, by expressing the intracellular domain of theNotch protein in the MSCs. MSCs are obtained by selecting adherent cells(i.e., cells that adhere to tissue culture plastic) from bone marrow.

Exemplary disclosures of MSCs are provided in U.S. patent applicationpublication No. 2003/0003090; Prockop (1997) Science 276:71-74 and Jiang(2002) Nature 418:41-49. Methods for the isolation and purification ofMSCs can be found, for example, in U.S. Pat. No. 5,486,359; Pittenger etal. (1999) Science 284:143-147 and Dezawa et al. (2001) Eur. J.Neurosci. 14:1771-1776. Human MSCs are commercially available (e.g.,BioWhittaker, Walkersville, Md.) or can be obtained from donors by,e.g., bone marrow aspiration, followed by selection for adherent bonemarrow cells. See, e.g., WO 2005/100552.

MSCs can also be isolated from umbilical cord blood. See, for example,Campagnoli et al. (2001) Blood 98:2396-2402; Erices et al. (2000) Br. J.Haematol. 109:235-242 and Hou et al. (2003) Int. J. Hematol. 78:256-261.Additional sources of MSCs include, for example, menstrual blood andplacenta.

Notch Intracellular Domain

The Notch protein is a transmembrane receptor, found in all metazoans,that influences cell differentiation through intracellular signaling.Contact of the Notch extracellular domain with a Notch ligand (e.g.,Delta, Serrate, Jagged) results in two proteolytic cleavages of theNotch protein, the second of which is catalyzed by γ-secretase andreleases the Notch intracellular domain (NICD) into the cytoplasm. Inthe mouse Notch protein, this cleavage occurs between amino acidsg1y1743 and val1744. The NICD translocates to the nucleus, where it actsas a transcription factor, recruiting additional transcriptionalregulatory proteins (e.g., MAM, histone acetylases) to relievetranscriptional repression of various target genes (e.g., Hes 1).

Additional details and information regarding Notch signaling are found,for example in Artavanis-Tsakonas et al. (1995) Science 268:225-232;Mumm and Kopan (2000) Develop. Biol. 228:151-165 and Ehebauer et al.(2006) Sci. STKE 2006 (364), cm7. [DOI: 10.1126/stke.3642006cm7].

Cell Culture and Transfection

Standard methods for cell culture are known in the art. See, forexample, R. I. Freshney “Culture of Animal Cells: A Manual of BasicTechnique,” Fifth Edition, Wiley, N.Y., 2005.

Methods for introduction of exogenous DNA into cells (i.e.,transfection), and methods for selection of cells comprising exogenousDNA, are also well-known in the art. See, for example, Sambrook et al.“Molecular Cloning: A Laboratory Manual,” Third Edition, Cold SpringHarbor Laboratory Press, 2001; Ausubel et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, New York, 1987 and periodicupdates.

SB623 Cells

In one embodiment for the preparation of SB623 cells, a culture of MSCsis contacted with a polynucleotide comprising sequences encoding a Notchintracellular domain (NICD); e.g., by transfection; followed byenrichment of transfected cells by drug selection and further culture.See, for example, U.S. Pat. No. 7,682,825 (Mar. 23, 2010); U.S. PatentApplication Publication No. 2010/0266554 (Oct. 21, 2010); and WO2009/023251 (Feb. 19, 2009); all of which disclosures are incorporatedby reference, in their entireties, for the purposes of describingisolation of marrow adherent stem cells and conversion of marrowadherent stem cells to SB623 cells (denoted “neural precursor cells” and“neural regenerating cells” in those documents).

In these methods, any polynucleotide encoding a Notch intracellulardomain (e.g., vector) can be used, and any method for the selection andenrichment of transfected cells can be used. For example, in certainembodiments, MSCs are transfected with a vector containing sequencesencoding a Notch intracellular domain and also containing sequencesencoding a drug resistance marker (e.g. resistance to G418). Inadditional embodiments, two vectors, one containing sequences encoding aNotch intracellular domain and the other containing sequences encoding adrug resistance marker, are used for transfection of MSCs. In theseembodiments, selection is achieved, after transfection of a cell culturewith the vector or vectors, by adding a selective agent (e.g., G418) tothe cell culture in an amount sufficient to kill cells that do notcomprise the vector but spare cells that do. Absence of selectionentails removal of said selective agent or reduction of itsconcentration to a level that does not kill cells that do not comprisethe vector. Following selection (e.g., for seven days) the selectiveagent is removed and the cells are further cultured (e.g., for twopassages).

Preparation of SB623 cells thus involves transient expression of anexogenous Notch intracellular domain in a MSC. To this end, MSCs can betransfected with a vector comprising sequences encoding a Notchintracellular domain wherein said sequences do not encode a full-lengthNotch protein. All such sequences are well known and readily availableto those of skill in the art. For example, Del Amo et al. (1993)Genomics 15:259-264 present the complete amino acid sequences of themouse Notch protein; while Mumm and Kopan (2000) Devel. Biol.228:151-165 provide the amino acid sequence, from mouse Notch protein,surrounding the so-called S3 cleavage site which releases theintracellular domain. Taken together, these references provide theskilled artisan with each and every peptide containing a Notchintracellular domain that is not the full-length Notch protein; therebyalso providing the skilled artisan with every polynucleotide comprisingsequences encoding a Notch intracellular domain that does not encode afull-length Notch protein. The foregoing documents (Del Amo and Mumm)are incorporated by reference in their entireties for the purpose ofdisclosing the amino acid sequence of the full-length Notch protein andthe amino acid sequence of the Notch intracellular domain, respectively.

Similar information is available for Notch proteins and nucleic acidsfrom additional species, including rat, Xenopus, Drosophila and human.See, for example, Weinmaster et al. (1991) Development 113:199-205;Schroeter et al. (1998) Nature 393:382-386; NCBI Reference Sequence No.NM_017167 (and references cited therein); SwissProt P46531 (andreferences cited therein); SwissProt Q01705 (and references citedtherein); and GenBank CAB40733 (and references cited therein). Theforegoing references are incorporated by reference in their entiretiesfor the purpose of disclosing the amino acid sequence of the full-lengthNotch protein and the amino acid sequence of the Notch intracellulardomain in a number of different species.

In additional embodiments, SB623 cells are prepared by introducing, intoMSCs, a nucleic acid comprising sequences encoding a Notch intracellulardomain such that the MSCs do not express exogenous Notch extracellulardomain. Such can be accomplished, for example, by transfecting MSCs witha vector comprising sequences encoding a Notch intracellular domainwherein said sequences do not encode a full-length Notch protein

Additional details on the preparation of SB623 cells, and methods formaking cells with properties similar to those of SB623 cells which canbe used in the methods disclosed herein, are found in U.S. Pat. Nos.7,682,825; 8,133,725; and U.S. Patent Application Publication Nos.2010/0266554 and 2011/0229442; the disclosures of which are incorporatedby reference herein for the purposes of providing additional details on,and alternative methods for the preparation of, SB623 cells, and forproviding methods for making cells with properties similar to those ofSB623 cells. See also Dezawa et al. (2004) J. Clin. Invest.113:1701-1710.

Reversal of Symptoms of TBI by Transplantation of SB623 Cells

The efficacy of SB623 cell transplantation as a treatment for TBI wastested in a rat model system. Prior to testing, adult maleSprague-Dawley rats (8-weeks old) were evaluated in motor andneurological tests (all performed by two investigators blinded to thetreatment condition throughout the study) to confirm that all animalsdisplayed normal behaviors at baseline (i.e., prior to brain insult).Animals were then exposed to experimental traumatic brain injury (TBI),and seven days later were subjected to the same behavioral tests toconfirm the typical TBI-induced motor and neurological impairments.Following these tests (at 7 days post-TBI), the animals were assignedrandomly to one of two groups to receive either stereotaxic transplantsof Notch-induced bone marrow-derived stem cells (SB623 cells)^(26,29) orvehicle infusion into the cortex (see Example 3).

The inventors have found that, at both one month and three monthspost-TBI, traumatically injured animals that received transplants ofSB623 cells displayed significantly improved motor and neurologicalfunctions, coupled with significantly reduced damage to the corticalcore and peri-injured cortical areas, compared to traumatically injuredanimals that received vehicle only (see Examples). These behavioral andphysical improvements were achieved with modest graft survival of 0.60%and 0.16% at one month and three months post-TBI, respectively. Othersites in the brain that are affected by TBI include the striatum and thehippocampus; hence transplantation of SB623 cells to the striatum andthe hippocampus can also be used for treatment of TBI affecting theseareas. In summary, transplantation of SB623 cells into brain-injuredanimals provided robust functional recovery despite lack of graftpersistence.

Creation of a Biobridge by Transplantation of SB623 Cells

Examination of host tissue in brain-injured animals that had receivedtransplants of SB623 cells, at one month post-TBI, revealed a surge ofendogenous cell proliferation (detected by Ki67 expression) anddifferentiation of neurogenic cells (detected by expression of nestin)in the peri-injured cortical areas and subventricular zone (SVZ). Astream of cells (expressing doublecortin) migrating along the corpuscallosum (CC) of these animals was also detected. In contrast, animalssubjected to experimental TBI that received vehicle alone displayedlimited cell proliferation, little neural differentiation, and onlyscattered migration in the peri-injured cortical areas. In addition,very few newly-formed cells were visible in the sub-ventricular zone ofthese control animals (see Examples).

At three months post-TBI, the brains from animals that had receivedSB623 cell transplants exhibited much higher levels of cellproliferation and neural differentiation encasing the peri-injuredcortical areas, along with a solid stream of neuronal cells (expressingboth nestin and doublecortin) migrating not just along but across the CCfrom the SVZ to the impacted cortex. Brains from injured animals thathad received only vehicle exhibited much more elevated levels of cellproliferation at three months post-TBI than at one month post-TBI, butthe newly-formed cells appeared “trapped” within the SVZ and the corpuscallosum; with only a small number of cells able to reach the impactedcortex. Quantitative analysis of Ki67, nestin and doublecortinimmunoreactivity in the SVZ, the CC, and the injured cortical areaindicated that the differences in expression of these markers, betweenanimals receiving SB623 cell transplants and animals receiving onlyvehicle, were statistically significant.

In a separate experiment, the biobridge formed by endogenous cellsmigrating from the SVZ to the site of injury was isolated by lasercapture microdissection (Espina et al. (2006) Nature Protoc. 1:586-603)and its zymogenic properties were analyzed. In this experiment, threegroups of animals were analyzed: (1) animals subjected to TBI followedby transplantation of SB623 cells at 7 days post-TBI, (2) animalssubjected to TBI followed by infusion of vehicle at 7 days post-TBI, and(3) control sham-operated age-matched adult Sprague-Dawley rats (n=3 pergroup). Zymographic assays of the laser-captured biobridges from animalssubjected to TBI revealed two-fold and nine-fold upregulation of matrixmetalloproteinase 9 (MMP-9) expression/activity in animals that receivedSB623 cell transplants, compared to vehicle-infused animals orsham-operated animals, at one month and three monthspost-transplantation, respectively (Example 11).

MMPs have been implicated in recovery in chronic brain injury²⁹, andinhibition of MMP activity has been shown to abrogate migration ofneurogenic cells from the SVZ into damaged tissues and to retardneurovascular remodeling³⁰. MMPs may thus play a role in facilitatinghost cell migration towards injured brain areas as part of the processby which SB623 cells provide functional recovery from TBI.

In summary, the inventors have discovered that transplantation of SB623cells remodeled the traumatically injured brain by creating a biobridgebetween the SVZ and the peri-injured cortex. This method of cell therapycan now be used to create similar biobridges between neurogenic andnon-neurogenic sites, to facilitate injury-specific migration of cellsacross tissues that might otherwise pose barriers to cell motility.

Formulations, Kits and Routes of Administration

Therapeutic compositions comprising SB623 cells as disclosed herein arealso provided. Such compositions typically comprise the SB623 cells anda pharmaceutically acceptable carrier. Supplementary active compoundscan also be incorporated into SB623 cell compositions.

The therapeutic compositions disclosed herein are useful for, interalia, treating TBI and modulating stem cell migration in the brain.Accordingly, a “therapeutically effective amount” of a compositioncomprising SB623 cells is any amount that reduces symptoms of TBI orthat stimulates migration of stem cells in the brain. For example,dosage amounts can vary from about 100; 500; 1,000; 2,500; 5,000; 10,000; 20,000; 50,000; 100,000; 300,000; 500,000; 1,000,000; 5,000,000 to10,000,000 cells or more (or any integral value therebetween); with afrequency of administration of, e.g., once per day, twice per week, onceper week, twice per month, once per month, depending upon, e.g., bodyweight, route of administration, severity of disease, etc. Thus, atherapeutically effective amount can comprise a plurality ofadministrations of the same amount, or different amounts, of SB623cells. In certain embodiments, a single administration of SB623 cells isa therapeutically effective amount.

Various pharmaceutical compositions and techniques for their preparationand use are known to those of skill in the art in light of the presentdisclosure. For a detailed listing of suitable pharmacologicalcompositions and techniques for their administration one may refer totexts such as Remington's Pharmaceutical Sciences, 17th ed. 1985;Brunton et al., “Goodman and Gilman's The Pharmacological Basis ofTherapeutics,” McGraw-Hill, 2005; University of the Sciences inPhiladelphia (eds.), “Remington: The Science and Practice of Pharmacy,”Lippincott Williams & Wilkins, 2005; and University of the Sciences inPhiladelphia (eds.), “Remington: The Principles of Pharmacy Practice,”Lippincott Williams & Wilkins, 2008.

The cells described herein may be suspended in a physiologicallycompatible carrier for transplantation. As used herein, the term“physiologically compatible carrier” refers to a carrier that iscompatible with the SB623 cells and with any other ingredients of theformulation, and is not deleterious to the recipient thereof. Those ofskill in the art are familiar with physiologically compatible carriers.Examples of suitable carriers include cell culture medium (e.g., Eagle'sminimal essential medium), phosphate buffered saline, Hank's balancedsalt solution+/−glucose (HBSS), and multiple electrolyte solutions suchas, e.g., Plasma-Lyte™ A (Baxter).

The volume of a SB623 cell suspension administered to a subject willvary depending on the site of transplantation, treatment goal and numberof cells in solution. Typically the amount of cells administered will bea therapeutically effective amount. As used herein, a “therapeuticallyeffective amount” or “effective amount” refers to the number oftransplanted cells which are required to effect treatment of theparticular disorder; i.e., to produce a reduction in the amount and/orseverity of the symptoms associated with that disorder. For example, inthe case of TBI, transplantation of a therapeutically effective amountof SB623 cells results in reduction and/or reversal of the symptoms ofTBI; e.g., restoration of locomotor activity and neurologicalperformance, and stimulation of migration of host neurogenic cells.Therapeutically effective amounts vary with the type and extent of braindamage, and can also vary depending on the overall condition of thesubject.

The disclosed therapeutic compositions can also include pharmaceuticallyacceptable materials, compositions or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent or encapsulating material, i.e.,carriers. These carriers can, for example, stabilize the SB623 cellsand/or facilitate the survival of the SB623 cells in the body. Eachcarrier should be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the subject.Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Exemplary formulations include, but are not limited to, those suitablefor parenteral administration, e.g., intrapulmonary, intravenous,intra-arterial, intra-ocular, intra-cranial, sub-meningial, orsubcutaneous administration, including formulations encapsulated inmicelles, liposomes or drug-release capsules (active agents incorporatedwithin a biocompatible coating designed for slow-release); ingestibleformulations; formulations for topical use, such as eye drops, creams,ointments and gels; and other formulations such as inhalants, aerosolsand sprays. The dosage of the compositions of the disclosure will varyaccording to the extent and severity of the need for treatment, theactivity of the administered composition, the general health of thesubject, and other considerations well known to the skilled artisan.

In additional embodiments, the compositions described herein aredelivered intracranially at or near a site of traumatic brain injury.Such localized delivery allows for the delivery of the compositionnon-systemically, thereby reducing the body burden of the composition ascompared to systemic delivery. Local delivery can be achieved, forexample, by intra-cranial injection, or through the use of variousmedically implanted devices including, but not limited to, stents andcatheters, or can be achieved by inhalation, phlebotomy, or surgery.Methods for coating, implanting, embedding, and otherwise attachingdesired agents to medical devices such as stents and catheters areestablished in the art and contemplated herein.

Another aspect of the present disclosure relates to kits for carryingout the administration of SB623 cells, optionally in combination withanother therapeutic agent, to a subject. In one embodiment, a kitcomprises a composition of SB623 cells, formulated in a pharmaceuticalcarrier, suitable for transplantation.

EXAMPLES

In the studies disclosed herein, rats were subjected to experimentaltraumatic brain injury (TBI) and, seven days later, those havingsufficient locomotor and neurological deficits received transplants ofeither SB623 cells or vehicle to the injured area. Values of locomotorand neurological performance were evaluated prior to TBI (baselinevalues), again at 7 days after TBI (prior to transplantation), andmonthly thereafter for 3 months after TBI.

Following completion of behavioral testing at 1 month and 3 months afterTBI, randomly selected animals were euthanized (n=10 per group) bytranscardial perfusion with 4% paraformaldehyde. Their brains wereremoved and sectioned for evaluation of persistence of the transplantedcells, histological appearance of brain tissue in and around the injuredarea, expression of various neural markers in and around the injuredarea, and zymogenic activity in and around the injured area.

Transplant outcomes were evaluated using the following criteria: 1)locomotor behavior via elevated body swing test (EBST) and Rotorod; (2)neurological performance via a Bederson-modified neurologicalexamination; 3) lesion volume via histology (H&E stained sections); 4)graft survival via immunohistochemistry using an antibody (HuNu) thatspecifically detects human cells, and; 5) mechanism-basedimmunohistochemial analyses of neuroprotection and/or regeneration usingantibodies directed against the grafted human cells and host cells.

Example 1: Preparation of MSCs and SB623 Cells

Bone marrow aspirates from adult human donors were obtained from LonzaWalkersville, Inc. (Walkersville, Md.) and plated in α-MEM (Mediatech,Herndon, Va.) supplemented with 10% fetal bovine serum (Hyclone, Logan,Utah), 2 mM L-glutamine (Invitrogen, Carlsbad, Calif.) andpenicillin/streptomycin (Invitrogen). Cells were cultured for three daysat 37° C. and 5% CO₂, to obtain a monolayer of adherent cells. Afterremoval of non-adherent cells, culture was continued under the sameconditions for two weeks. During this time, cells were passaged twice,using 0.25% trypsin/EDTA. A portion of the cells from the second passagewere frozen as MSCs.

The remaining cells from the second passage were plated and transfected,using Fugene6 (Roche Diagnostics, Indianapolis, Ind.), with a plasmidcontaining sequences encoding a Notch intracellular domain operativelylinked to a cytomegalovirus promoter (pCMV-hNICD1-SV40-Neo^(R)). Thisplasmid also contained sequences encoding resistance to neomycin andG418 under the transcriptional control of a SV40 promoter. Transfectedcells were cultured at 37° C. and 5% CO₂ in the growth medium describedin the previous paragraph, supplemented with 100 μg/ml G418 (Invitrogen,Carlsbad, Calif.). After seven days, G418-resistant colonies wereexpanded and the culture was passaged twice. After the second passage,the cells were collected and frozen as SB623 cells.

In an embodiment, preparation of cells for use in inducing proliferationand migration of endogenous neurogenic cells, referred to below as“neural regenerating cells” (NRCs), are prepared by the followingmethod.

Preparation of Marrow Adherent Stromal Cells (MASCs)

Bone marrow aspirates, obtained from human donors, were divided into12.5 ml aliquots in 50 ml tubes, and 12.5 ml of growth medium (10% FBSin α-MEM, supplemented with penicillin/streptomycin and 2 mML-glutamine) was added to each tube. The contents of the tubes weremixed by inversion and the tubes were centrifuged at 200×g for 8minutes. The upper, clear phase was discarded, the volume of the lowerphase was adjusted to 25 ml with fresh growth medium, and the tubes wereagain mixed and centrifuged. The upper layer was again removed. Thevolume of the lower phase in each tube was again adjusted to 25 ml andthe contents of all tubes was pooled in a 250 ml tube. Afterdetermination of cell concentration by Trypan Blue exclusion anddetermination of nucleated cell count, cells were plated in T225 flasks,in 40 ml per flask of growth medium at a density of 100×10⁶ totalnucleated cells per flask. The flasks were incubated at 37° C. for 3days in a CO₂ incubator, during which time the MASCs attached to theflask.

After 3 days, unattached cells were removed by rocking the flasks andwithdrawing the culture medium. Each flask was washed three times with40 ml of α-MEM supplemented with penicillin/streptomycin; then 40 ml ofprewarmed (37° C.) growth medium was added to each flask and the cellswere cultured at 37° C. in a CO₂ incubator. During this time, the mediumwas replaced with 40 ml of fresh growth medium every 3-4 days, and cellswere monitored for growth of colonies and cell density.

When the cultures achieved 25-30% confluence (usually 10,000-20,000cells per colony and within 10-14 days), the MASCs (passage MO) wereharvested for further passage. MASCs were harvested from up to 10 T-225flasks at a time. Medium was removed from the flasks and the adherentcells were rinsed with 20 ml of DPBS w/o Ca/Mg (DPBS−/−, HyClone) 2times. Ten ml of 0.25% Trypsin/EDTA (Invitrogen, Carlsbad, Calif.) wasadded to each flask and flasks were incubated for approximately 5 min atroom temperature. When cells had detached and the colonies had dispersedinto single cells, the trypsin was inactivated by addition of 10 ml ofgrowth medium followed by gentle mixing. The cell suspensions werewithdrawn from the flasks, and pooled in 250 ml tubes. The tubes weresubjected to centrifugation at 200×g for 8 minutes. The supernatantswere carefully removed and the wet cell pellets were resuspended ingrowth medium to an estimated cell concentration of approximately 1×10⁶cells/ml. Viable cell count was determined and cells were plated in T225flasks at a concentration of 2×10⁶ cells per flask in growth medium(passage M1). Cells were grown for 3-5 days, or until 85-90% confluent,changing medium every 2 to 3 days. At 85-90% confluence, passage M1cells were harvested by trypsinization and replated at 2×10⁶ cells perT225flask as described above, to generate passage M2 cultures. M2cultures were fed fresh medium every three days, if necessary. Whenpassage M2 cultures reached 85-90% confluence (usually within 3-5 days),they were either harvested for transfection to generate NRCs (see below)or frozen for future use.

Preparation of Neural Regenerating Cells (NRCs)

Neural regenerating cells were made by transfection of passage M2 MASCswith a plasmid encoding the Notch intracellular domain. The plasmid(pCI-Notch) comprised a pCI-neo backbone (Promega, Madison, Wis.) inwhich sequences encoding amino acids 1703-2504 of the human Notch-1protein, which encode the intracellular domain, were introduced into themultiple cloning site. For each flask of MASCs, 5 ml of transfectionmixture, containing 40 μg of plasmid and 0.2 ml of Fugene 6® solution,was used. To make the transfection mixture, the appropriate amount ofFugene® solution (depending on the number of flasks of cells to betransfected) was added to α-MEM in a sterile 250 ml tube, using a glasspipette. The solution was mixed gently and incubated for 5 min at roomtemperature. The appropriate amount of plasmid DNA was then addeddropwise to the Fugene®/α-MEM mixture, gently mixed, and incubated for30 min at room temperature.

Prior to the addition of pCI-Notch DNA to the Fugene®/MEM mixture, 5 mlwas removed and placed into a 15 ml tube to which was added 40 ug ofpEGFP plasmid. This solution was used to transfect one flask of cells,as a control for transfection efficiency.

For transfection, passage M2 MASCs were harvested by trypsinization (asdescribed above) and plated at a density of 2.5×10⁶ cells in 40 ml ofgrowth medium per T225 flask. When the cells reached 50-70% confluence(usually within 18-24 hours) they were prepared for transfection, byreplacing their growth medium with 35 ml per flask of transfectionmedium (α-MEM+10% FBS without penicillin/streptomycin).

Three hours after introduction of transfection medium, 5 ml of thetransfection mixture (as described above) was added to each T-225 flaskby pipetting directly into the medium, without contacting the growthsurface, followed by gentle mixing. A control T-225 flask wastransfected with 40 μg of pEGFP plasmid, for determination oftransfection efficiency.

After incubating cultures at 37° C. in transfection medium for 24 hours,the transfection medium was replaced with α-MEM+10%FBS+penicillin/streptomycin.

Cells that had incorporated plasmid DNA were selected 48 hrs aftertransfection by replacing the medium with 40 ml per flask of selectionmedium (growth medium containing 100 μg/ml G-418). Fresh selectionmedium was provided 3 days, and again 5 days after selection was begun.After 7 days, selection medium was removed and the cells were fed with40 ml of growth medium. The cultures were then grown for about 3 weeks(range 18 to 21 days), being re-fed with fresh growth medium every 2-3days.

Approximately 3 weeks after selection was begun, when surviving cellsbegan to form colonies, cells were harvested. Medium was removed fromthe flasks using an aspirating pipette and 20 ml of DPBS withoutCa²⁺/Mg²⁺, at room temperature, was added to each flask. The culturesurface was gently rinsed, the wash solution was removed by aspirationand the rinse step was repeated. Then 10 ml of prewarmed (37° C.) 0.25%Trypsin/EDTA was added to each flask, rinsed over the growth surface,and the flasks were incubated for 5-10 min. at room temperature.Cultures were monitored with a microscope to ensure complete detachmentof cells. When detachment was complete, trypsin was inactivated byaddition of 10 ml of growth medium per flask. The mixture was rinsedover the culture surface, mixed by pipetting 4-5 times with a 10 mlpipette, and the suspension was transferred into a sterile 50 ml conicalcentrifuge tube. Cells harvested from several flasks could be pooled ina single tube. If any clumps were present, they were allowed to settleand the suspension was removed to a fresh tube.

The cell suspensions were centrifuged at 200×g for 8 min at roomtemperature. Supernatants were removed by aspiration. Cell pellets wereloosened by tapping the tube, about 10 ml of DPBS without Ca²⁺/Mg²⁺ wasadded to each tube and cells were resuspended by gently pipetting 4-5times with a 10 ml pipette to obtain a uniform suspension.

For expansion of transfected cells, cell number was determined for thesuspension of transformed, selected cells and the cells were plated inT-225 flasks at 2×10⁶ cells per flask (providing approximately 30%seeding of viable cells). This culture is denoted M2P1 (passage #1).M2P1 cultures were fed with fresh medium every 2-3 days, and when cellsreached 90-95% confluence (usually 4-7 days after passage), they wereharvested and replated at 2×10⁶ cells per flask to generate passageM2P2. When M2P2 cultures reached 90-95% confluence, they were harvestedfor cryopreservation or for further assay.

Cryopreservation

MASCs and NRCs were frozen for storage according to the followingprocedure. MASCs were typically frozen after passage M2, and NRCs weretypically frozen after passage M2P2. Processing 4-5 flasks at a time,medium was aspirated from the culture flasks, 10 ml of 0.25%Trypsin/EDTA (at room temperature) was added to each flask, gentlyrinsed over the culture surface for no longer than 30 sec, and removedby aspirating. Then 10 ml of warmed (37° C.) 0.25% Trypsin/EDTA wasadded to each flask, rinsed over the growth surface, and the flasks wereincubated for 5-10 min. at room temperature. Cultures were monitored bymicroscopic examination to ensure complete detachment of cells.

When detachment was complete, 10 ml of α-MEM growth medium was added toeach flask, rinsed over the culture surface, and detached cells weremixed by pipetting 4-5 times with a 10 ml pipette. The cell suspensionwas transferred into a sterile 250 ml conical centrifuge tube, and anylarge clumps of cells were removed. Cells harvested from 15-20 flaskswere pooled into one 250 ml tube.

The tube was subjected to centrifugation at 200×g for 8 min at roomtemperature. The supernatant was removed by aspirating. The pellet wasloosened by tapping the tube, and about 25 ml of DPBS (−/−) was added toeach tube. Cells were resuspended by gently pipetting 4-5 times with a10 ml pipette to obtain a uniform suspension. Any clumps in thesuspension were removed by pipetting each sample through a sterile 70 μmsieve placed in the neck of a 50 ml tube.

Cell suspensions were pooled in a 250 ml centrifuge tube and anyremaining clumps were removed. The final volume was adjusted to 200 mlwith DPBS (−/−) and the sample was subjected to centrifugation at 200×gfor 8 min at room temperature. The supernatant was removed byaspiration. The cell pellet was loosened by tapping, 20 ml of DPBS (−/−)was added to the tube and cells were resuspended by mixing well andgently pipetting with a 10 ml pipette. The final volume was adjustedwith DPBS (−/−) to give an estimated concentration of approximately0.5-1.0×10⁶ cells/ml, usually about 4-5 ml per T225 flask harvested, orabout 200 ml for a 40-flask harvest. A viable cell count was conductedon the suspension, which was then subjected to centrifugation at 200×gfor 8 minutes. The supernatant was aspirated, and the cell pellet wasresuspended in cold Cryo Stor solution (BioLife Solutions, Bothell,Wash.) to a concentration of 12×10⁶ cells/ml. One ml aliquots weredispensed into vials, which were sealed and placed at 4° C. in a CryoCooler. Vials were transferred into a CryoMed (Thermo Forma) freezerrack and frozen.

MSCs and SB623 cells prepared as described herein were thawed asrequired and used for further study.

Example 2: Induction of TBI in a Rat Model

A total of 40 animals identified at baseline (prior to TBI surgery) asexhibiting normal behaviors (50-60% bias swing activity in EBST; 60seconds staying time on Rotorod; and a mean Bederson score of at most0-0.5), received TBI surgery as described below.

All surgical procedures were conducted under aseptic conditions. Adultmale Sprague-Dawley rats were anesthetized with 1.5% isofluorane andchecked for pain reflexes. Under deep anesthesia, animals underwent amoderate TBI model, as follows. Each animal was placed in a stereotaxicframe, with anesthesia being maintained with 1-2% isofluoraneadministered via a gas mask. After exposing the skull, a 4-mmcraniectomy was performed over the left frontoparietal cortex, with itscenter at −2.0 mm AP and +2.0 mm ML to the bregma. A pneumaticallyoperated metal impactor, with a diameter of 3 mm, was used to impact thebrain at a velocity of 6.0 m/s, reaching a depth of 1.0 mm below thedura mater layer and remaining in the brain for 150 milliseconds. Theimpactor rod was angled 15° to the vertical, so as to be perpendicularto the tangential plane of the brain surface at the impact site. Alinear variable displacement transducer (Macrosensors, Pennsauken, N.J.)connected to the impactor was used to measure velocity and duration, toverify consistency.

Subsequent to controlled cortical impact injury, the incision wassutured after bleeding ceased. An integrated heating pad and rectalthermometer unit with feedback control allowed maintenance of bodytemperature at normal limits. All animals were monitored until recoveryfrom anesthesia. In addition, animals were weighed and observed dailyfor three consecutive days following induction of TBI, weighed twice aweek thereafter, and monitored daily throughout the study for healthstatus and any signs that indicated problems or complications.

Example 3: Grafting of SB623 Cells

Of the animals subjected to TBI, only those having the following degreeof behavioral impairment at Day 7 post-TBI were selected fortransplantation studies: at least 75% bias swing activity in the EBST;30 seconds or less staying time on the Rotorod; and a mean Bedersonscore of at least 2.5. Those animals that were selected were randomlyassigned either to a group receiving SB623 transplants (n=20) or to agroup receiving vehicle infusion (n=20). The target area fortransplantation was the medial cortex, which corresponded to theperi-injured cortical area based on previously established target sitesfor similar stereotaxic implants.

All surgical procedures were conducted under aseptic conditions. Animalswere anesthetized with 1.5% isofluorane and checked for pain reflexes.Once deep anesthesia was achieved (as determined by the loss of painreflex), the hair was shaved around the area of the surgical incision(skull area), leaving enough border to prevent contamination of theoperative site. This was followed by two surgical germicidal scrubs ofthe site, and draping with sterile drapes.

The animal was then fixed to a stereotaxic apparatus (Kopf Instruments,Tujunga, Calif.), and a small opening was made in the skull with a burr.The coordinates of the opening were 0.5 mm anterior and 1.0 mm lateralto the bregma and 2.0 mm below the dural surface; these were selected tocorrespond to the cortical area adjacent to the core injury site, basedon the atlas of Paxinos and Watson (1998). A 26-gauge Hamilton syringe,containing test material, was then lowered into the opening. With asingle needle pass, 3 deposits of 3 ul each were made. Each depositconsisted of 100,000 viable cells in 3 ul of Plasmalyte A, infused overa period of 3 minutes. Following an additional 2-minute absorption time,the needle was retracted and the wound was closed with a stainless steelwound clip. A heating pad and a rectal thermometer allowed maintenanceof body temperature at about 37° C. throughout surgery and followingrecovery from anesthesia. Control injections contained Plasmalyte Aonly.

Treated and control animals were subjected to elevated body swing test(EBST, Example 4), neurological examination (Example 5), and the Rotorodtest (Example 6) at baseline (prior to TBI), at 7 days after TBI (justprior to transplantation) and monthly thereafter up to 3 monthspost-TBI.

In addition, brains of treated and control animals were characterizedhistologically at one and three months post-TBI to determine degree ofdamage (Examples 8 and 9); the extent of proliferation, migration andneural differentiation of host cells (Example 10); and the presence ofzymogenic activity (Example 11).

Example 4: Elevated Body Swing Test (EBST)

All investigators testing the animals were blinded to the treatmentcondition. The EBST was conducted by handling the animal by its tail andrecording the direction in which the animal swung its head. The testapparatus consisted of a clear Plexiglas box (40×40×35.5 cm). The animalwas gently picked up at the base of the tail, and elevated by the tailuntil the animal's nose was at a height of 2 inches (5 cm) above thesurface. The direction of the swing (left or right) was recorded oncethe animal's head moved sideways approximately 10 degrees from themidline position of the body. After a single swing, the animal wasplaced back in the Plexiglas box and allowed to move freely for 30seconds prior to retesting. These steps were repeated for a total of 20assays for each animal. Uninjured rats display a 50% swing bias, thatis, the same number of swings to the left and to the right. A 75% swingbias indicated 15 swings in one direction and 5 in the other during 20trials. Previous results utilizing the EBST have indicated thatunilaterally lesioned animals display>75% biased swing activity at onemonth after a nigrostriatal lesion or unilateral hemispheric injury; andthat such asymmetry is stable for up to six months^(3,26).

The results of the EBST are shown in FIG. 1. After TBI, essentially allanimals exhibited biased swing activity. In animals transplanted withSB623 cells, biased swing activity steadily decreased over thethree-month period following TBI and transplantation. By contrast, inanimals transplanted with vehicle, the percentage of animals exhibitingbiased swing activity after TBI remained essentially unchanged.

Example 5: Modified Bederson Neurological Examination

About one hour after conclusion of the EBST, a modifiedBederson-Neurological exam was conducted, following the procedurespreviously described^(3,26) with minor modifications. Neurologic scorefor each rat was obtained using 3 tests which included (1) forelimbretraction, which measured the ability of the animal to replace theforelimb after it was displaced laterally by 2 to 3 cm, graded from 0(immediate replacement) to 3 (replacement after several seconds or noreplacement); (2) beam walking ability, graded 0 for a rat that readilytraversed a 2.4-cm-wide, 80-cm-long beam to 3 for a rat unable to stayon the beam for 10 seconds; and (3) bilateral forepaw grasp, whichmeasured the ability to hold onto a 2-mm-diameter steel rod, graded 0for a rat with normal forepaw grasping behavior to 3 for a rat unable tograsp with the forepaws. The scores from all 3 tests, which wereconducted over a period of about 15 minutes on each assessment day, wereadded to give a mean neurologic deficit score (maximum possible score, 9points divided by 3 tests=3).

The results of these neurological examinations are shown in FIG. 2.After TBI, the mean neurological score was 2.5 (out of 3) in allanimals. In animals transplanted with SB623 cells, this score wassteadily reduced (indicating improved neurological function) over thethree-month period following TBI and transplantation. Improvement ofneurological function in animals transplanted with SB623 cells wasstatistically significant (p<0.05) compared to animals that had beeninfused with vehicle.

Example 6: Rotorod® Test

One hour after completion of the neurological exam, the animals weresubjected to the Rotorod® test. This test involved placement of theanimal on a rotating treadmill that accelerates from 4 rpm to 40 rpmover a 60-second period (Rotorod®, Accuscan, Inc., Columbus, Ohio). Thetotal number of seconds an animal was able to remain on the treadmillwas recorded and used as an index of motor coordination. Previousresults using a TBI model system have shown that injured animals wereable to remain on the Rotorod for significantly shorter times, comparedto sham-operated or normal controls.

The results of this assay are shown in FIG. 3. Uninjured animals wereable to remain on the treadmill for an average of 60 seconds. The meantime on the treadmill fell to below 20 seconds seven days after TBI. Inanimals transplanted with SB623 cells after TBI, mean time on thetreadmill doubled to approximately 40 seconds. These improvements werestatistically significant compared to animals subjected to TBI that hadbeen infused with vehicle.

Example 7: Perfusion and Sectioning

At 1 month and 3 months after TBI, following completion of behavioraltesting as described in Examples 4-6, randomly-selected rats wereeuthanized (n=10 per group) by transcardial perfusion with 4%paraformaldehyde. The brains were dissected, post-fixed overnight in 4%paraformaldehyde, then immersed in 30% sucrose. Beginning at bregma-5.2mm anteriorly, each forebrain was cut into 40 um coronal sections,moving posteriorly until bregma-8.8 mm. Sections were processed fordeterminations of brain damage and analysis of cell survival in theperi-lesion area as described in Examples 8 and 9.

Example 8; Measurement of Brain Damage

Preparation and examination of brain sections was undertaken to identifythe extent of brain damage and host cell survival. At least 4 coronaltissue sections per brain were processed for hematoxylin and eosin (H&E)or Nissl staining Cerebral damage was quantitated by determining theindirect lesion area, which was calculated by subtracting the intactarea of the ipsilateral hemisphere from the area of the contralateralhemisphere. The lesion volume was presented as a volume percentage ofthe lesion compared to the contralateral hemisphere, by summing lesionareas from serial sections.

The results, shown quantitatively in FIG. 4B, indicate that animalssubjected to TBI that received transplants of SB623 cells experiencedsignificantly less damage to the cortical core and the peri-injuredcortical areas, compared to animals subjected to TBI that receivedinfusion of vehicle.

Example 9: Analysis of Cell Survival in the Peri-TBI Lesion Area

Randomly selected high power fields, corresponding to the peri-injuredcortical area, were examined to count surviving host cells in thisregion. Results are shown in FIG. 4A.

Example 10: Immunohistochemistry

Floating sections were processed for immunofluorescent microscopy.Briefly, 40 μm cryostat sectioned tissues were examined at 4×magnification and digitized using a PC-based Image Tools computerprogram. Engraftment of transplanted SB623 cells was assessed usingmonoclonal human specific antibody HuNu that did not cross-react withrodent proteins. Additional brain sections were processed formechanism-based immunohistochemical analyses of brain tissue samplesfocusing on cell proliferation (Ki67), migration (doublecortin or DCX)and neural differentiation (nestin). Brain sections were blind-coded andAbercrombie's formula was used to calculate the total number ofimmunopositive cells^(3,26).

The results of these analyses showed that transplantation of SB623 cellsinduced formation of a biobridge between the SVZ and the impacted cortexconsisting of highly proliferative, neurally committed and migratorycells. At one month post-TBI, immunofluorescent and confocal microscopyrevealed a surge of endogenous cell proliferation (evidenced by cellsexpressing Ki67) and immature neural differentiation (cells expressingnestin) in the peri-injured cortical areas and subventricular zone(SVZ), with a stream of migrating cells (cells expressing doublecortin)along the corpus callosum (CC) of the animals that had receivedtransplants of SB623 cells. Brains from animals that had receivedvehicle alone displayed limited cell proliferation and neuraldifferentiation, and scattered migration in the peri-injured corticalareas, with almost no newly formed cells present in the SVZ. At threemonths post-TBI, the brains from SB623-transplanted animals exhibitedmuch more massive cell proliferation and neural differentiation encasingthe peri-injured cortical areas, accompanied by a solid stream ofneuronally labeled cells (expressing both nestin and doublecortin)migrating, not just along, but across the CC from the SVZ to theimpacted cortex. By contrast, in brains from vehicle-infused animals,cell proliferation was enhanced, but the newly formed cells were“trapped” within the SVZ and the CC and only a few cells were able toreach the impacted cortex. Quantitative analyses of Ki67, nestin and DCXimmunolabeled cells in SVZ, CC and CTX revealed statisticallysignificant differences between transplanted and vehicle-infused animals(FIGS. 5-7).

Example 11: Zymography

A separate cohort of animals from that whose analysis was described inExamples 4-10 was used to test for the presence and/or activity ofproteolytic enzymes after transplantation of SB623 cells into injuredbrain. Rats were subjected to TBI, then transplanted with either SB623cells or vehicle. A control group of age-matched sham-operated adultSprague-Dawley rats was subjected to the same experimental procedure(n=3 rats per group). At one month and three months after TBI, tissuecorresponding to the biobridge formed by the cells migrating from theSVZ to the impacted cortex was obtained by laser dissection. Afterextraction, the tissue was placed in cryotubes and flash frozen inliquid nitrogen. The tubes were stored in a −80° C. freezer untilhomogenization.

Samples were homogenized in 450 μL of cold working buffer containing 50mM Tris-HCl (pH 7.5), 75 mM NaCl, and 1 mM PMSF. The tissue wasprocessed with a homogenizer for 10 minutes and centrifuged at 4° C. for20 minutes at 13000 rpm. The supernatants were separated, frozen andkept at −80° C. until use. The total protein concentration in thesupernatant was assessed by the Bradford method.

On the day that zymography was conducted, a volume equivalent to 50 μgof total protein was loaded into a freshly prepared gelatin zymographygel. All gels contained a control lane that was loaded with 0.5 ngrecombinant MMP-9, which was used as a standard for both enzyme amount(in ng) and gelatinolytic activity (expressed as relative opticaldensity units, see below). Proteins were electrophoretically separatedin the gel under non-reducing conditions at 100 V. After electrophoresisthe gels were washed in 125 ml 2.5% Triton twice for 20 minutes. Thegels were then incubated in activation buffer (Zymogram DevelopmentBuffer, Bio-Rad, Hercules, Calif.) for 20 hours at 37° C. The next day,the gels were stained with Coomassie Blue R-250 Staining Solution(Bio-Rad) for 3 hours and destained for 25 minutes with Destain Solution(Bio-Rad). The gelatinolytic activity of the samples was assessed bydensitometric analysis (Gel-Pro v 3.1, Media Cybernetics, Carlsbad,Calif.) of the bands. The molecular weights of proteins in regions ofthe gel exhibiting lytic activity were determined by comparison topre-stained standard protein marker (Bio-Rad) run on the same gel.Activity was expressed as optical density relative to that of 0.5 ng ofrecombinant MMP-9, which was run in the gel as a standard.

The results are shown in FIG. 8. The laser-captured biobridges(corresponding to brain tissue between the SVZ and the impacted cortex)from animals transplanted with SB623 cells after TBI expressed highlevels of MMP-9 gelatinolytic activity at one month and three monthspost-TBI. The levels in SB623-treated animals were significantly higherthan those in biobridges from vehicle-infused and sham-operated animals(p<0.05) at both time points. Although biobridges from vehicle-infusedanimals showed a significant increase in MMP-9 activity compared tosham-operated animals at one month post-TBI, these levels reverted tocontrol levels (i.e., not significantly different from those ofsham-operated animals) at three months post-TBI.

For detection on blots, membranes were blocked with blotting gradenon-fat dry milk (Bio-Rad). After washing with 0.1% tween20-tris-buffered saline (TTBS), the membranes were incubated with 1ug/ml anti MMP-9 monoclonal mouse antibody overnight at 4° C. Membraneswere washed again in TTBS, incubated with secondary antibody (1:1,000dilution of horseradish peroxidase-conjugated goat anti-mouse IgG,Calbiochem) for one hour and finally developed with horseradishperoxidase development solution (ECL advance detection kit, Amersham).The membranes were exposed to autoradiography films (Hyblot CL, DenvilleScientific Inc.). The density of the sample bands for the zymograms wasexpressed as maximal optical density relative to the standard band (0.5ng recombinant MMP-9).

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We claim:
 1. A method for inducing migration of neurogenic cells from aneurogenic niche to a site of brain injury in a subject, the methodcomprising: introducing cells into the brain between the neurogenicniche and the site of brain injury, wherein said cells are obtained by amethod comprising: (a) providing a culture of marrow adherent stem cells(MSCs); (b) contacting the culture of (a) with a polynucleotidecomprising sequences encoding a Notch intracellular domain (NICD); (c)selecting cells that comprise the polynucleotide of (b); (d) withdrawingselection; and (e) further culturing the cells in the absence ofselection.
 2. The method of claim 1, wherein the brain injury is atraumatic brain injury.
 3. The method of claim 2, wherein the traumaticbrain injury results from external mechanical force.
 4. The method ofclaim 1, wherein the subject is a mammal.
 5. The method of claim 4,wherein the subject is a human.
 6. The method of claim 1, wherein theneurogenic niche is the subventricular zone.
 7. The method of claim 1,wherein the site of brain injury is in the cortex.
 8. The method ofclaim 1, wherein the MSCs are obtained from a human.
 9. A method forinducing proliferation of neurogenic cells in the brain of a subject,the method comprising: introducing cells into the brain between aneurogenic niche and a site of brain injury, wherein said cells areobtained by a method comprising: (a) providing a culture of marrowadherent stem cells (MSCs); (b) contacting the culture of (a) with apolynucleotide comprising sequences encoding a Notch intracellulardomain (NICD); (c) selecting cells that comprise the polynucleotide of(b); (d) withdrawing selection; and (e) further culturing the cells inthe absence of selection.
 10. The method of claim 9, wherein the subjecthas a traumatic brain injury.
 11. The method of claim 10, wherein thetraumatic brain injury results from external mechanical force.
 12. Themethod of claim 9, wherein the subject is a mammal.
 13. The method ofclaim 12, wherein the subject is a human.
 14. The method of claim 9,wherein the neurogenic cells reside in the subventricular zone.
 15. Themethod of claim 9, wherein the neurogenic cells migrate to the cortex.16. The method of claim 1, wherein the MSCs are obtained from a human.